Shear Stress-induced [Ca2+]i Transients and Oscillations in Mouse Fibroblasts Are Mediated by Endogenously Released ATP

Abstract

The effects of ATP, U-73122, apyrase, and saline shear stress on [Ca2+]i homeostasis were studied in fura-2 loaded, mouse fibroblast cells (L929), both in suspension and plated on glass. Release of internal Ca2+ was induced by ATP, via a receptor identified pharmacologically as a P2U type. In single cells, low concentrations of ATP evoked [Ca2+]i oscillations. These events were blocked by the putative phospholipase C inhibitor, U-73122 (but not by the inactive analog U-73343) and by the ATP/ADPase, apyrase. In addition, both these agents reduced the [Ca2+]i of unstimulated cells, especially after stirring, and blocked spontaneously occurring [Ca2+]i oscillations, which suggested an already activated state of the ATP receptor, independent from exogenous stimulations. Moreover, it was found that stirring of the cells was correlated with a steady accumulation of inositol phosphates, also blockable by apyrase, and that [Ca2+]i mobilization could be induced by puffs of saline in single cells. The transition to a Ca(2+)-free environment also provoked [Ca2+]i oscillations, most likely via the increase in ATP4- concentration. This evidence suggests that endogenous ATP is released from L fibroblasts in response to fluid shear stress, and this results in an autocrine, tonic up-regulation of the phosphoinositide signaling system and an ensuing alteration in Ca2+ homeostasis. Up until now, such a response to shear stress was believed to be unique to endothelial cells.

P2 receptor-mediated signaling in mast cell biology

“…Mechanic stimulation of mouse fibroblasts, hepatocytes, or glial cells evokes an increase in Ca 2+ not only in stimulated cells but also in distant cells [100][101][102]. This remote signaling is mediated by ATP or other nucleotides.…”

The touching story of purinergic signaling in epithelial and endothelial cells

“…The P2X 4 receptor is the highest expressed P2 receptor in endothelium [79,80]. By using antisense oligonucleotides, the P2X 4 receptor was shown to be important for shear stress-dependent Ca 2+ influx via an ATP-dependent mechanism [81].…”

“…Diadenosine polyphosphates such as AP 4 A, AP 5 A, and AP 6 A are combinations of two adenosine molecules connected with four to six phosphate groups. They have been identified as vasocontractile agents [89], probably via actions on P2X 1 and P2Y 2 receptors.…”

“…AP 5 A and AP 6 A are stored at higher levels in platelets from patients with hypertension and may contribute to their increased peripheral vascular resistance [90]. UP 4 A is a novel endotheliumderived vasoconstrictive factor more potent than endothelin in renal vasoconstriction [91]. It is released upon stimulation of the endothelium by acetylcholine, thrombin, and mechanical stress and can be cleaved into either ATP or UTP to stimulate both P2X 1 and P2Y 2 receptors on VSMC resulting in increased blood pressure [91].…”

“…cells to mechanical stimulation, a phenomenon that also has been scientifically documented [3][4][5][6][7]. Renal epithelial cells, especially Madin-Darby canine kidney (MDCK) cells, a well-differentiated cell line derived from distal tubule/collecting duct, was found to respond to nucleotide signaling via P2 receptor activation already 30 years ago [8].…”